α‐Hederin induces paraptosis by targeting GPCRs to activate Ca2+/MAPK signaling pathway in colorectal cancer

Abstract Background Non‐apoptotic cell death is presently emerging as a potential direction to overcome the apoptosis resistance of cancer cells. In the current study, a natural plant agent α‐hederin (α‐hed) induces caspase‐independent paraptotic modes of cell death. Purpose The present study is aimed to investigate the role of α‐hed induces paraptosis and the associated mechanism of it. Methods The cell proliferation was detected by CCK‐8. The cytoplasm organelles were observed under electron microscope. Calcium (Ca2+) level was detected by flow cytometry. Swiss Target Prediction tool analyzed the potential molecule targets of α‐hed. Molecular docking methods were used to evaluate binding abilities of α‐hed with targets. The expressions of genes and proteins were analyzed by RT‐qPCR, western blotting, immunofluorescence, and immunohistochemistry. Xenograft models in nude mice were established to evaluate the anticancer effects in vivo. Results α‐hed exerted significant cytotoxicity against a panel of CRC cell lines by inhibiting proliferation. Besides, it induced cytoplasmic vacuolation in all CRC cells. Electron microscopy images showed the aberrant dilation of endoplasmic reticulum and mitochondria. Both mRNA and protein expressions of Alg‐2 interacting proteinX (Alix), the marker of paraptosis, were inhibited by α‐hed. Besides, both Swiss prediction and molecular docking showed that the structure of α‐hed could tightly target to GPCRs. GPCRs were reported to activate the phospholipase C (PLC)‐β3/ inositol 1,4,5‐trisphosphate receptor (IP3R)/ Ca2+/ protein kinase C alpha (PKCα) pathway, and we then found all proteins and mRNA expressions of PLCβ3, IP3R, and PKCα were increased by α‐hed. After blocking the GPCR signaling, α‐hed could not elevate Ca2+ level and showed less CRC cell cytotoxicity. MAPK cascade is the symbol of paraptosis, and we then demonstrated that α‐hed activated MAPK cascade by elevating Ca2+ flux. Since non‐apoptotic cell death is presently emerging as a potential direction to overcome chemo‐drug resistance, we then found α‐hed also induced paraptosis in 5‐fluorouracil‐resistant (5‐FU‐R) CRC cells, and it reduced the growth of 5‐FU‐R CRC xenografts. Conclusions Collectively, our findings proved α‐hed as a promising candidate for inducing non‐apoptotic cell death, paraptosis. It may overcome the resistance of apoptotic‐based chemo‐resistance in CRC.


| INTRODUCTION
Colorectal cancer (CRC) incidence and mortality is rapidly growing worldwide. 1The advancements made in the treatment of CRC, mainly include surgery and chemotherapy.5-fluorouracil (5-FU)-based chemotherapeutic regimens are the conventional treatments for CRC, but it is usually not highly effective against metastatic colorectal cancer (mCRC), and resistance of chemotherapy will accelerate the progression of CRC.Thus, it is crucial to discover novel strategies for CRC treatment.
Cell death is a fundamental cellular process.In certain circumstances, cells regulate their death in a programmed dependent way.Apoptosis is the classical form of programmed cell death. 2 Inhibition or evasion of apoptosis is partially characterized by CRC, 3 which in turn endows tumors advantage for survival and contributes to anticancer therapy resistance. 4Therefore, characterization of non-apoptotic cell death may suggest strategies that could offer alternatives to anti-cancer therapeutic approaches.
6][7][8] The feature of paraptosis is accompanied by massive cytoplasmic vacuolation, 9 alterations in organelle structure, and enlargement of both the endoplasmic reticulum (ER) and mitochondria. 10The regulatory pathways of paraptosis encompass mitogen-activated protein kinases (MAPKs) and its modulation is inversely associated with the activity of Alg-2 interacting proteinX (Alix). 11araptosis was reported to occur in various cancer cells that were treated with natural anti-cancer agents. 5urcuminoid B63 has been shown to elicit paraptosis in gastric cancer cells through ROS-induced ER stress coupled with MAPK pathway activation. 12Similarly, Jolkinolide B incites both paraptosis and apoptosis in bladder cancer cells through ROS-driven ER stress mechanisms. 8Furthermore, in non-small cell lung cancer cells, celastrol has been identified as an inducer of paraptosis to overcome resistance to afatinib. 13-Hederin (α-hed) is a chemical compounds that can be extracted by Akebia trifoliata (Thunb.)Koidz plants.It has gained attention for its anti-cancer effects in recent years.14,15 The present study shows that αhed induced paraptosis-like cell death, the proparaptotic activity of αhed in CRC cells was mediated through targeting G-protein-coupled receptors (GPCRs) to activate phospholipase C (PLC)-β3, PLCβ3 produced signals to stimulate calcium (Ca 2+ ) signaling by activation of inositol 1,4,5-trisphosphate receptor (IP3R), which subsequently activated protein kinase C alpha (PKCα) and led to the MAPK activation.Interestingly, αhed was effective against chemotherapy-resistant CRC both in vivo and in vitro.Our results suggest that αhed may provide an alternative therapeutic strategy for CRC.

| Cell lines and reagents
CRC cell lines, including LoVo, HT29, and HCT-116, were sourced from the American Type Culture Collection.To establish stable 5-FU resistant CRC cells, HT-29 cell line were treated with gradually increasing concentration of 5-FU (from 10 to 300 μM initially), cells were passaged three times at each 5-FU concentration of 10, 20, 40, 80, 150, 300 μM.We assessed 5-FU resistance at each dose by calculating the IC50 using CCK8 assays.These lines were propagated in DMEM medium from KeyGen Biotechn, Nanjing, China, which was enriched with 10% fetal bovine serum and 1% penicillin-streptomycin, both acquired from Gibco, Eggenstein, Germany.Meanwhile, αhederin was procured from Chengdu Herbpurify CO., LTD (purity>98%), the chemical structure of αhederin is shown in Figure S1.α-Hederin was initially dissolved in DMSO before being further diluted using the aforementioned DMEM medium.

| Cell viability assay
For CRC cell viability analysis, 5 × 10 3 cells were dispensed into each compartment of a 96-well format.Subsequently, the cells were exposed to varying doses of αhed ranging from 0 to 56 μM.In another setting, a consistent concentration of 24 μM αhed was administered for distinct time intervals: 0, 6, 12, 24, 48, and 72 h.Post-treatment, the CCK-8 assay from Beyotime, Nantong, China, was utilized to determine cell viability.Absorbance readings were procured at a wavelength of 450 nm using instrumentation from Tecan Group Ltd., Männedorf, Switzerland.

| Transmission electron microscopy
Exposure of HT-29 cells to DMSO served as the vehicle control, whereas another set experienced 16 μM αhed intervention for a duration of 24 h.Subsequent procedures involved fixing these cells in 2.5% glutaraldehyde, maintained at 4°C over a 12-h period.This was followed by post-fixation using 1% OsO₄ for an additional hour, staining processes with uranyl acetate, and a dehydration step.The final embedding was done using the Poly/ Bed 812 resin, sourced from Pelco.Detailed cellular observations were carried out with an electron microscope, specifically the FEI Tecnai G2 from Thermo Scientific, USA.

| Reverse transcription and quantitative real-time PCR
For the synthesis of cDNA, total RNA underwent reverse transcription utilizing the PrimeScript RT Reagent Kit, a product from Vazyme, Nanjing, China.The quantification process for Alix, PLCβ3, IP3R, and PKC was carried out with their expression levels adjusted based on GAPDH.The primers specified in subsequent sections were employed for the RT-qPCR execution (Table 1).

GPCRs structural model
Molecular docking methods were used to evaluate binding abilities of αhed with GPCRs.The 2D structure of αhed was downloaded from PubChem database, and then transformed into 3D structure with mimimum structural energy by ChemBio3D program.3D structures of the GPCRs were retrieved from PDB database, and dehydrated by PyMOL 2.5.2.Then, AutoDockTools 1.5.6 performed molecular docking of αhed with GPCRs.PyMOL 2.5.2 was used to visualize the binding modes of αhed and GPCRs.

| CRC xenografts
The Institutional and Local Committee on the Care and Use of Animals at Nanjing University of Chinese T A B L E 1 List of primers.

Genes
Forward Reverse Medicine granted permission for all described experiments (Ethical approval number, 202110A025).Nude male mice, 5 weeks old and weighing between 18 and 22 g, were procured from Vital River Laboratories, Beijing, China.These mice (N = 30) were distributed randomly across five distinct groups.Each mouse received a subcutaneous injection on their right flank with HT-29 cells, where the injection volume was 100 μL PBS containing 1 × 10 6 cells.Following a period of 14 days post-injection, when tumors approached an approximate volume of 100 mm 3 , a regimen of treatments commenced.The mice underwent intraperitoneal injections either with 5-FU at a dose of 25 mg/kg or αhed at dosages of 1.5, 1, or 0.5 mg/kg, administered biweekly.This treatment spanned 21 days.On Days 7, 14, 21, 28, and 35, tumor dimensions, including length and width, were recorded.The volume was then computed using the formula V = 0.5lw 2 .Concluding the experiments on the 35th day, the mice underwent euthanasia, post which tumor tissues were extracted and subsequently weighed.

| Tissue staining
Tumor samples underwent fixation in a 10% formalin solution prior to being embedded in paraffin.Subsequent sectioning yielded slices with a thickness of 3 μm.For the purpose of observing histopathological changes, staining was conducted using hematoxylin and eosin (H&E).The immunohistochemical (IHC) methodology implemented included the use of primary antibodies targeting Alix and ki-67, both at a dilution ratio of 1:200.
Post incubation with the respective secondary antibody, visualization of positive staining was achieved through the application of diaminobenzidine (DAB).Moreover, the TUNEL assay, relying on a colorimetric approach, was executed in accordance with guidelines provided by Beyotime.

| Statistical analysis
For the purposes of data analysis, the GraphPad Prism version 8 platform was employed.Utilizing one-way ANOVA, variations of significance were assessed.A threshold of p < 0.05 served as the criterion for deeming differences to hold statistical significance.

| α-Hed inhibits cell viability and induces cytoplasmic vacuolation in CRC cells
In assessing the potential anti-tumor capabilities of αhed against CRC cells, three distinct CRC cell strains (LOVO, HT-29, and HCT-116) and one normal human colon epithelial cell (NCM-460) underwent treatment with varying αhed concentrations for a 24-h period, followed by viability testing using the CCK-8 method.A marked inhibition in cell proliferation was observed across all strains, manifesting in a dose-responsive manner.The corresponding IC50 values were approximately 48 μM for LOVO, 23 μM for HT-29, and 19 μM for HCT-116, 40 μM for NCM-460, as indicated in Figure 1A.When exposed to 24 μM of αhed, a time-sensitive suppression of proliferation was apparent in each cell strain.The viability was reduced by half at around 14 h for HCT-116, 24 h for HT-29, 46 h for LOVO, and 48 h for NCM-460 (Figure 1B).Interestingly, cytoplasmic vacuolation was observed in LOVO, HT-29, and HCT-116 cells upon αhed exposure (Figure 1C).Examination via electron microscopy highlighted deviations in the morphology of both ER and mitochondria.Specifically, cells subjected to αhed demonstrated pronounced ER swelling, coupled with subtle mitochondrial enlargement (Figure 1D).Previous literature has underscored the role of Alix downregulation in F I G U R E 1 α-Hed inhibits cell viability and induces cytoplasmic vacuolation in CRC cells.(A) CRC cell lines (LOVO, HT-29, HCT-116) and human colon epithelial cell (NCM-460) underwent treatment across varying αhed concentrations (ranging from 0 to 56 μM) over a span of 24 h, followed by the assessment of cell viability through the CCK-8 assay method.(B) CRC cell lines (LOVO, HT-29, HCT-116) and human colon epithelial cell (NCM-460) were exposed to a consistent αhed concentration of 24 μM over varied time intervals (0, 6, 12, 24, 48, 72 h).Subsequent cell viability was evaluated using the CCK-8 assay.(C) Phase contrast microscopy observations were made on LOVO, HT-29, and HCT-116 cells after 24 h of exposure to αhed at concentrations of 24, 12, and 10 μM respectively, [scale bar =50 μm].(D) Following a 24 h treatment with 16 μM αhed, HT-29 cell morphological changes were inspected using electron microscopy, revealing mitochondrial and ER swelling post-treatment, [Magnification: either ×10,000 or 30,000, scale bars at 2 μm or 500 nm].(E, F) Western blot investigations were conducted for Alix expression.For LOVO cells, the treatment was categorized into three dosages: low at 12 μM (α-hed-L), medium at 24 μM (α-hed-M), and high at 48 μM (α-hed-H).HT-29 cells were exposed to 6, 12, and 24 μM, while HCT-116 cells had treatments of 5, 10, and 20 μM for the same categories.GAPDH served as the loading reference, with densitometric quantifications performed via ImageJ.(G) Alix's expression levels were assessed using RT-qPCR.The categorizations for LOVO, HT-29, and HCT-116 remain aligned with the prior description.GAPDH functioned as the loading control.Significant differences are denoted as *p < 0.05, **p < 0.01 in comparison with the control group.augmenting paraptosis, 11,16 suggesting that Alix functions as a potent indicator for paraptosis.In line with this, both protein and mRNA concentrations of Alix in HT-29 cells treated with αhed were seen to reduce in a dose-sensitive manner (Figure 1E-G).
However, αhed may also induce the caspase-dependent apoptosis in CRC cells.We have conducted western blot analysis of cleaved caspase-3 in HT-29 cells at 24, 48 and 72 h after treatment with αhed (Figure S2A,B).The results showed that αhed activated the cleaved caspase3 expression in CRC cells.To determine if apoptosis is the decisive form of αhed induced cell death, we inhibited apoptosis by a caspase inhibitor, Z-VAD fluoromethyl ketone (Z-VAD-FMK). 17Previous study has shown that 5-FU induces caspase-dependent apoptosis, 18 and it could be blocked by Z-VAD-FMK, 19 so we used 5-FU as a positive control to induces apoptosis.Our study showed that both αhed and 5-FU increased the expression of cleaved caspase3, however, Z-VAD-FMK can't rescue the elevated cleaved caspase3 expression that induced by αhed.In contrast, Z-VAD-FMK rescued the elevated cleaved caspase3 expression that induced by 5-FU (Figure S2C,D).These results indicated that apoptosis is not the only main form of cell death following αhed exposure, it would support the induction of paraptosis may exert decisive influence to CRC cell death.
GPCRs are links of extracellular signals to the control of cell fate, such as survival, proliferation and death. 20ome downstream regulators of GPCRs, such as PLCβ3, IP3R, PKCα were able to ignite Ca 2+ signaling. 21CRC Cells were treated with 12 μM αhed for the indicated time points, at 6 h of αhed treatment, the protein and mRNA expression levels of PLCβ3 were markedly increased.Coincidentally, IP3R started to elevate at 6 h.At 20 h of αhed treatment, expression of both IP3R and PKCα were significantly increased (Figure 2C-E).CCG258747, a GPCR kinases (GRKs) subfamily-selective inhibitor, was used to impair efficacy on GPCRsdependent process in cells. 22We subsequently explored if CCG258747 could block the activation of PLCβ3/IP3/ Ca 2+ /PKCα pathway by αhed.CCG258747, given alone, did not significantly modify the expression of PLCβ3, IP3R, PKCα, but upregulated Alix.It significantly diminished the promoting effects of αhed on both the proteins and mRNA expression of PLCβ3, IP3R, PKCα while counteracted the inhibitory effect of αhed on Alix (Figure 2F-H), confirming the presumption that αhed targets GPCRs to activate PLCβ3/IP3R/PKCα signaling pathway.

Ca 2+ levels are critical for αhed-induced paraptosis
Cytosolic Ca 2+ signals mediate diverse cellular responses, it generated through the coordinated translocation of Ca 2+ across the ER membrane.ER and mitochondria are the main reservoirs of Ca 2+ . 23We found both ER and mitochondria swelled after αhed exposure, dysregulation of ion homeostasis may result in abnormal osmotic pressure, which in turn lead to morphological changes of organelles, so we presumed that αhed may disrupt the concentration of cytosolic Ca 2+ level.
Utilizing flow cytometry and the cell-permeable Ca 2+indicator dye, Fluo-3, it was observed that when HT-29 cells underwent treatment with αhed, there was a pronounced enhancement in the levels of intracellular Ca 2+ .This augmentation was noted to initiate around the 10-h mark, escalating sharply by 14 h, and culminating in a peak at the 24-h interval post the administration of αhed (Figure 3A,B).Interestingly, CCG258747 significantly diminished the promoting effects of αhed on Fluo-3 fluorescence intensity (Figure 3C), confirming the presumption that αhed targets GPCRs to elevate intracellular Ca 2+ levels.We then observed the localization of Ca 2+ to the ER, mitochondria.As shown in Figure S4, the untreated HT-29 cells exhibited evenly distributed Ca 2+ , and no obvious localization commonality with ER or mitochondria was observed.After treatment with αhed, the Fluo-3 fluorescence intensity increased significantly, and aggregated in clusters, which adjacent to ER-tracker red, coinciding with mitochondrialtracker-red.These results indicate that αhed elevated intracellular Ca 2+ levels, and the release of Ca 2+ from ER is subsequently uptaken by mitochondria.
Subsequently, the research focus shifted to identifying potential evidence that suggests the role of Ca 2+ in facilitating the paraptosis triggered by αhed.We tested the effect of the SEA0400, a specific Na + /Ca 2+ exchange inhibitor, 24 on αhed induced cytotoxicity.As shown in Figure 3D, SEA0400 effectively prevented vacuolization formation in αhed-treated cells.Besides, it also blocked the inhibitory effect of αhed treatment in HT-29 cells viability, while counteracted the promoting effect of αhed on cell death.(Figure 3E,F).Via Western blot analysis, when HT-29 cells underwent concurrent treatment with SEA0400, the diminished Alix protein and mRNA levels caused by αhed were reverted to normal (Figure 3G-I).
The data gathered implies the significant elevation in intracellular Ca 2+ levels is related to GPCRs signaling.This study probed the phosphorylation status of JNK, ERK, and p38 to evaluate MAPK activation dynamics.Results from Western blotting revealed that while αhed exposure to HT-29 cells had minimal effect on JNK, ERK, and p38 expression, was a notable augmentation in the phosphorylation of MAPK proteins, demonstrating a dose-responsive relationship (Figure 4A,B).Moreover, concurrent treatment with SEA0400 in HT-29 cells thwarted the αhedtriggered phosphorylation states of JNK, ERK, and p38 (Figure 4C,D).Such findings substantiate the crucial role of MAPK signaling cascade in the Ca 2+ driven paraptosis in CRC cells.

CRC cell line by αhed may be effective compared to chemotherapeutic agents
Most chemotherapeutic agents, such as 5-FU, exert inhibitory activity in cancer by inducing apoptosis, thus non-apoptotic cell death may suggest strategies that could offer alternatives to chemo-resistant CRC therapeutic approaches.We then determined whether αhed, which induces paraptosis, is able to target resistant CRC cancer cells.To test this hypothesis, we set up 5-FU-resistant CRC clones by a step-wise dose escalation study as reported previously. 26Cell viability in the presence of 5-FU clearly showed that derived HT-29 clones are resistant to 5-FU, the IC50 of 5-FU-resistant and parental HT-29 clones are being at approximately 200, 75 μM respectively (Figure 5A).Remarkably, reduction of viability by exposure of αhed in 5-FU-resistant and parental HT-29 cells showed barely difference (Figure 5B).αhed also induced cytoplasmic vacuolation in 5-FU resistant HT-29 cells, indicating initiation of paraptosis-like features (Figure 5C).Besides, αhed also decreased expression of Alix in 5-FU-resistant HT-29 cells in a does-dependent manner (Figure 5D).Collectively, αhed may effectively inhibit apoptosis-based 5-FU-resistant CRC cells via inducing paraptosis-like cell death.

| α-Hed inhibits 5-FU-R HT-29 xenograft growth in mice
In order to validate whether αhed possesses inhibitory effects on chemo-resistant CRC in vivo, a study was conducted in nude mice with chemo-resistant HT-29 tumor xenografts to assess the therapeutic potential.
These mice underwent treatment via intraperitoneal injection of either 5-FU (25 mg/kg) or varying dosages of αhed (0.5, 1, 1.5 mg/kg).Observations indicated that 5-FU administration barely led to a suppression in CRC progression relative to the control.Furthermore, intermediate αhed doses (0.5, 1 mg/kg) exhibited marginal tumor growth deceleration.The dose of 1.5 mg/kg of αhed marked a significant decline in tumor volumes and weights (Figure 6A-C), which seemed to surpass the inhibitory capability of 5-FU.Analysis of excised tumor samples further elucidated αhed's impact on CRC expansion.Histological evaluation via H&E staining displayed cytoplasmic vacuolation, mirroring attributes of paraptosis-resembling cellular fatality (Figure 6D).Yet, it remains imperative to highlight observed fluctuations in body weight measurements across both 5-FU and αhed administered groups (Figure S5).
Through IHC staining, it became evident that administering αhed led to a notable suppression in cellular proliferation, as illustrated by the diminished expression of Ki67.Moreover, signs pointing towards paraptosis as the decreased expression of Alix (Figure 6E-H).In contrast, the PLCβ3/IP3R/PKCα pathway was increased (Figure S6).Additionally, cell death was witnessed (Figure S7).All these observations collectively hint that αhed induces paraptosis and contributes to an observable restraint on chemo-resistant CRC growth when assessed in vivo.

| DISCUSSION
Since cancer cells develop adaptive abilities to escape apoptosis, other forms of programmed cell death has emerged to overcome apoptotic-based drug resistance. 27ccumulating evidences have shown that compounds from natural plants has been an efficient alternative way in inventing anti-cancer drugs. 28In the current study, a natural plant substance αhed with pronounced cytotoxic efficacies to CRC cells was identified, it triggers paraptosis, F I G U R E 3 Elevated intracellular Ca 2+ levels are critical for αhed-induced paraptosis.(A, B) Fluo-3 stained HT-29 cells, post their exposure to 12 μM αhed across distinct time intervals (ranging from 0 to 24 h), underwent FACS evaluation.The graph on the left captures the fluorescence intensities (FI) for specified durations (0, 14, 24 h) of αhed treatment.Histogram to the right represents Fluo-3 FI post exposure to 22 μM αhed across 0-24 h.(C) HT-29 cells were treated with αhed (12 μM), CCG258747 (50 nM), αhed combined with CCG258747 respectively for 24 h, then stained with Fluo-3 and processed for FACS analysis.FI in cells treated with αhed for 0, 14, 24 h were denoted in the graph.Histogram to the right represents Fluo-3 FI in HT-29 cells treated with αhed (12 μM), CCG258747 (30 nM), αhed combined with CCG258747 respectively for 24 h is shown.(D) HT-29 cells were exposed to a combination of treatments, which included αhed (12 μM), the Na + /Ca 2+ exchange inhibitor SEA0400 (50 nM), or a blend of αhed and SEA0400, for a period of 24 h.These were then visualized using the phase contrast microscope [scale bar =50 μm].(E) HT-29 Cells, as grouped in prior panels, underwent evaluation of their viability via the CCK-8 assay.(F) HT-29 Cells, as grouped in prior panels, was ascertained by Annexin V/PI staining method.(G, H) Western blot investigations for Alix were conducted, with cells treated as described earlier.Densitometric quantification of immunoblots was processed through ImageJ.(I) RT-qPCR analysis of Alix was performed, with cell groupings maintained as previously detailed.Significance markers: *p < 0.05, **p < 0.01, ***p < 0.001, in relation to the control group.instead of apoptotic cell death in CRC cells.Here we show that αhed directly targeting multiple subunits of GPCRs to stimulate Ca 2+ release from ER stores, and subsequently activate the characteristic pathway of paraptosis, the MAPK cascade.Extensive cytoplasmic vacuolations are strongly associated with elevated intracellular Ca 2+ levels, osmotic pressure that induced by dysregulation of ion homeostasis may in turn lead to ER/mitochondrial dilation.Besides, the Na + /Ca 2+ exchange inhibitor, SEA0400 effectively prevented vacuolization formation in αhed-treated cells.Interestingly, Ca 2+ signaling has been highlighted to be detrimental to cancer cells, 29 and we found SEA0400 effectively block the cytotoxic effect of αhed treatment in HT-29 cells.As we presumed, αhed was effective against chemotherapy-resistant CRC both in vivo and in vitro.These findings suggest that αhed is a potential therapeutic candidate for CRC via a Ca 2+ -mediated paraptotic mechanism, the central players behind the anti-cancer activity were GPCRs-mediated downstream pathways.
GPCRs activation is universally fundamental for the regulation of cell functions. 30,31The GPCRs intracellular domain is bound to a heterotrimer of guanine nucleotide-binding proteins, comprising a GDP-bound F I G U R E 4 Evidence for the role of αhed in the initiation of the MAPK cascade via Ca 2+ pathways.(A, B) HT-29 cells underwent treatment with diverse concentrations of αhed, specifically αhed-L (6 μM), αhed-M (12 μM), and αhed-H (24 μM), sustained over a 24 h duration.This was followed by a Western blot assessment, examining the presence and phosphorylation states of JNK, ERK, and p38.Densitometric evaluations of the obtained immunoblots were facilitated using ImageJ software.(C, D) HT-29 cells were subjected to specific treatments: a concentration of 12 μM αhed, the Na + /Ca 2+ exchange inhibitor SEA0400 at 50 nM, and a combined treatment involving both αhed (12 μM) and SEA0400 (50 nM).A subsequent Western blot evaluation focused on determining the levels and phosphorylation statuses of JNK, ERK, and p38.ImageJ was once again employed for quantifying the densitometry of the produced immunoblots.Significance indicators: *p < 0.05, **p < 0.01 when juxtaposed with the control group.
Gα, Gβ Gγ subunits. 32Upon ligand binding, replacement of GDP with GTP, induces the dissociation of the heterotrimer, as well as the detachment of Gα from the Gβγ subunits.The liberated Gα and Gβγ dimers then evoke signaling pathways that mediate multiple cellular functions. 33,34Gα binds to and activates PLCβ enzymes, which hydrolyses the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2) into the IP3 and DAG, and this is ultimately followed by a transient rise in Ca 2+ level via release from ER stores. 35Four PLCβ isozymes (PLCβ1-4) share the same hydrolytic reaction, 36 of which PLCβ3 is elevated by αhed, and we also demonstrated its elevated protein expression after αhed exposure.PKC isozymes (α, β, γ) is a group of serine/threonine protein kinases that are physiologically activated by DAG and Ca 2+ , 37 it regulates cell death by transmitting extracellular signals to downstream pathways, MAPK is the one of the responding cascades. 38We then found protein expression of PKCα could be elevated after αhed exposure, but counteracted by CCG258747.Of note, we also found MAPK is activated by αhed, as the phosphorylated forms of ERK, p38, and JNK were all increased, and this effect could be diminished by SEA0400, supporting the presumption that αhed targets GPCRs to induce Ca 2+ mediated paraptosis-like cell death in CRC (Figure 7).
Therefore, αhed may render an alternative option to overcome apoptotic-based drug resistance by inducing paraptosis, and exploiting an opportunity for natural anticancer drug development.

| CONCLUSION
α-Hed promotes paraptosis-like cell death in CRC.The mechanism is facilitated by the surge in GPCRs mediated Ca 2+ signaling, which subsequently triggering MAPK cascade.Comprehensive evaluations further corroborate the efficacy of αhed in chemo-resistant CRC both in vivo and in vitro conditions.Data from this research hints at the possibility of considering αhed as a potential alternative approach in CRC therapy.

3. 4
| α-Hed activates MAPK cascade by Ca 2+ signaling Cytosolic Ca 2+ have been shown to trigger MAPK signaling, which has implications for paraptosis and vacuolation-related cell demise.

F
I G U R E 5 α-Hed overcomes chemotherapy resistance in CRC cells by induction of paraptosis.(A, B) Effects of 5-FU and αhed on HT-29 cell viability.Cells were developed resistant to 5-FU through a dose-escalation assay.Parental cells (WT) and 5-FU-resistant lines (5-FU-R) were then exposed to increasing concentrations of 5-FU (0, 40, 80, 120, 160, 200, 240, 280 μM) or αhed (0, 2, 4, 8, 16, 24, 32, 40, 48, 56 μM) for 24 h.(C) Phase images of 5-FU-R HT-29 cells were exposed to αhed-L (6 μM), αhed-M (12 μM), αhed-H (24 μM) for 24 h [scale bar =20 μm].(D) Western blot analysis of Alix.The grouping details are in consistent with above.ImageJ analyzed densitometric quantification of the immunoblots.*p < 0.05, **p < 0.01 compared to control group.| 13 of 15 RAO et al.F I G U R E 6 α-Hed inhibits 5-FU-R HT-29 xenograft growth in mice.(A) Visual documentation of excised tumor tissues obtained from the experimental mice.(B) Tracing the progression of tumor dimensions at specific intervals after the introduction of HT-29 cells into the nude mice.(C) The final evaluation of tumor mass at the termination of the experimental period.(D) Histological examination via H&E stain of the collected tumor samples.Noticeable αhed-triggered cytoplasmic vacuolation is highlighted with red pointers [scale bar = 100 μm].(E-H) IHC analysis of the tumor sections, concentrating on Ki-67 and Alix expression.The presence of immunoreactive substances was identified using the DAB chromogen, appearing in a brown hue [scale bar = 100 μm].F I G U R E 7 A schematic mechanism underlying αhederin induces calcium-mediated paraptosis-like cell death by targeting GPCRs in colorectal cancer (CRC).